PROJECT SUMMARY/ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is estimated to occur in one third of the general population and is a major predisposing factor in the pathogenesis of hepatic insulin resistance and type 2 diabetes (T2D). NAFLD occurs when lipid supply to the liver exceeds rates of lipid oxidation and lipid export. A number of therapies have been employed to reduce ectopic-lipid accumulation and hepatic insulin resistance, however these approaches have been met with limited success in the long-term and new drugs are required. The acetyl-CoA carboxylase (ACC) isoenzymes, ACC1 and ACC2, catalyze the synthesis of malonyl-coA, a precursor for fatty acid synthesis and an allosteric inhibitor of the carnitine/palmitoyl shuttle system for fatty acid oxidation. Given its unique position in intermediary metabolism, pharmalogic inhibition of ACC offers an attractive therapy to simultaneously inhibit fatty acid synthesis and stimulate fatty acid oxidation, favorable outcomes in treating obesity, diabetes and fatty liver disease. In this regard, our lab has previously demonstrated that antisense oligonucleotide-mediated inhibition of hepatic ACC1 and ACC2 results in marked reductions in hypertriglyceridemia, hepatic triglyceride content and reversal of hepatic insulin resistance in a high fat-fed rodent model of NAFLD. Here, we will evaluate the impact of a novel hepatospecific small molecule inhibitor of ACC1 and ACC2 (GS-834356) on hepatic steatosis and hepatic insulin resistance in rat models of diet-induced obesity. In addition, we will perform a comprehensive set of hepatic metabolic flux measurements to assess rates of hepatic mitochondrial oxidation, rates of anaplerosis, rates of ketogenesis, and hepatic de novo lipogenesis (DNL). We hypothesize that chronic inhibition of hepatic ACC by GS-834356 will lead to reduced hepatic fat content, due to increases in hepatic mitochondrial oxidation and reduced DNL, which in turn will lead to increased hepatic insulin sensitivity. [Recent preliminary data from our lab has also demonstrated that chronic inhibition of hepatic ACC by GS-834356 paradoxically drives hypertriglyceridemia. As such, we also aim to elucidate the molecular mechanism by which this is occurring. Collectively, the results of this research will provide valuable insight in the metabolic effects of liver-specific inhibition of ACC, which will improve our understanding of basic lipid biology and may lead to the development/improvement of pharmacologic approaches for treating NAFLD and T2D.]